The blaNDM-1 and mcr-1 genes coexist in Escherichia coli strain isolated from public trash cans

Carbapenem-resistant Enterobacteriaceae are among the most critical priority pathogens that pose a significant threat to human health. Within the Enterobacteriaceae family, Escherichia coli is the predominant bacterium. Colistin serves as a crucial ‘last line’ treatment for infections caused by carbapenem-resistant Enterobacteriaceae. But the coexistence of bla NDM-1 and mcr-1 in both Enterobacteriaceae and non-Enterobacteriaceae has become widespread on a global scale. The observed phenomenon is particularly noteworthy in E. coli isolated from clinical sources,

Carbapenem-resistant Enterobacteriaceae are among the most critical priority pathogens that pose a significant threat to human health.Within the Enterobacteriaceae family, Escherichia coli is the predominant bacterium.Colistin serves as a crucial 'last line' treatment for infections caused by carbapenem-resistant Enterobacteriaceae.But the coexistence of bla NDM-1 and mcr-1 in both Enterobacteriaceae and non-Enterobacteriaceae has become widespread on a global scale.The observed phenomenon is particularly noteworthy in E. coli isolated from clinical sources, 1 animal farming 2 and wastewater. 3It is also noteworthy in clinically isolated Klebsiella pneumoniae 4 and Pseudomonas aeruginosa. 5Furthermore, the majority of reported resistance due to these two genes in these strains is located on plasmids.Research on antimicrobial resistance genes (ARGs) in environmental health is crucial, especially in locations such as public trash cans where human waste is disposed.These sites may contain ARGs that pose risks of transmission to humans, either through airborne dissemination or direct contact with sanitation workers.Given the role of waste disposal in the urban ecosystem, these aspects warrant comprehensive investigation within the 'One Health' framework, which emphasizes the interconnectedness of human, animal and environmental health.This holistic approach is essential for understanding and mitigating the potential public health impacts associated with antimicrobial resistance.In this study, we found for the first time that bla NDM-1 and mcr-1 genes coexist in E. coli isolated from public trash cans and that mcr-1 is located on a plasmid but bla NDM-1 is located on the chromosome.
From December 2022 to April 2023, E. coli strain ECSD228 carrying bla NDM-1 and mcr-1 was isolated from 253 cotton swab samples collected from trash cans in Shandong Province.Concurrently, antimicrobial susceptibility testing was performed using a microbroth dilution method, and the breakpoints for each antimicrobial agent were determined according to CLSI standards.The strain ECSD228 was found to be resistant to both meropenem and colistin, with MIC values of 4 mg/L (Table S1, available as Supplementary data at JAC-AMR Online).Illumina NovaSeq 6000 and Oxford Nanopore GridION sequencing platforms were then used, followed by hybrid assembly using Unicycler v0.5.0.The results revealed that strain ECSD228 contained six plasmids, and ResFinder analysis predicted the presence of additional ARGs alongside bla NDM-1 and mcr-1 in strain ECSD228 (Table S2).MLST analysis demonstrated that ECSD228 belonged to ST617.
The WGS results revealed that mcr-1 was located on the 66 460 bp circular plasmid pE228-MCR-66K, with an average GC content of 43%.This plasmid belongs to the IncI2 type, which is known for its high prevalence in carrying the mcr-1 gene. 6The plasmid pE228-MCR-66K not only carries mcr-1 but also contains an additional ARG called bla CTX-M-55 .Comparative analysis using full plasmid BLAST query demonstrated that pE228-MCR-66K exhibits close similarity with other IncI2 plasmids.Specifically, pE228-MCR-66K shared 99.98% identity with pEC16-50-MCR, pM-199-C35, pMCR-M21015, pSH13G841, pSh487-m4 and pJD053-MCR59K; moreover, their coverage rates exceeded 90%, indicating a high likelihood of horizontal transfer of resistance genes among IncI2 plasmids. 7Additionally, pE228-MCR-66K contains the type IV secretion system (T4SS), which is conserved in all IncI2 plasmids and is responsible for plasmid transfer (Figure 1a).Further genetic analysis revealed that mcr-1 is integrated downstream of the nikB gene, and the genetic structure 'traL-mobC-nikA-nikB-mcr-1-pap2-traE' is highly similar to plasmids pJD053-MCR-59K, pSH15G1531 and pSh069-m6 of the plasmid type IncI2 (Table S3).The mcr-1-pap2 element has been horizontally transferred into a different plasmid skeleton, again indicating that mcr-1 has become widespread. 8However, compared with the original mcr-1 carrier plasmid (pHNSHP45) from China, the absence of the ISApl1 gene upstream of the mcr-1 gene increased the stability of mcr-1 in the plasmid

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(Figure 1b).Conjugative transfer plays a pivotal role in the spread of plasmid-borne resistance genes, posing a significant threat to public health.Conjugative transfer assays employed azide-resistant E. coli J53 as the recipient strain to confirm the transferability of pE228-MCR-66K carrying the mcr-1 gene.It was observed that despite mcr-1 being present on the plasmid pE228-MCR-66K, transfer to the recipient strain did not occur in this study.To further validate the potential transfer of the mcr-1 gene, we conducted natural transformation experiments to assess the ability of DNA fragments carrying the resistance gene to undergo natural transformation into recipient bacteria; however, no positive results were obtained (Figure S1).
There are a few reports that bla NDM-1 is located on chromosomes.In addition to bla NDM-1 , the strain ECSD228 also carried other ARGs: aadA16, sul1, dfrA27, arr-3 and aac(6′)-Ib-cr are located in an 11.7 kb multiple antimicrobial resistance region.This region contains a complex class 1 integron, along with a 5′ conserved segment (5′CS), two 3′CSs, a common region ISCR1, and two variable regions (VRs), components that in turn carry AMR genes.bla NDM-1 is embedded in VR-2 attached to the resistance gene arr-3 and catB3, VR-2 and 3′CS-2 linked by ISCR1, and can form a modular structure: 5′CS (intI1)-VR-1 (aac (6′)-Ib-cr, arr-3, dfrA27, aadA16)-3′CS-1 (qacE-sul1)-ISCR1-VR-2 (trpF, ble MBL , bla NDM-1 , catB3, arr-3)-3′CS-2 (qacE-sul1).In this study, the genetic context (ISCR1-trpF-ble MBL -bla NDM-1 -catB3-arr-3-qacE-sul1) of bla NDM-1 located on chromosomes showed high similarity with pCf75, pHD1688-NDM and pJNE2-NDM belonging to the IncA/C2 plasmid types.However, different from pRH-228, the downstream link of ISCR1 is not traF, but aphA6 and ISAba125.At the same time, there is deletion of three resistance genes, catB3, qacE and arr-3, between ble MBL and sul1 genes downstream of bla NDM-1 , and the deletion of arr-3, dfrA27 and aadA16 between aac(6′)-Ib-cr and qacE genes upstream of bla NDM-1 .This means that the ability of ISCR1-mediated class 1 integrons to capture different kinds of resistance genes in different bacteria is not completely consistent. 9The bla NDM-1 gene is primarily transmitted through plasmids, and the presence of insertional elements IS26, ISCR1 and IS6100 can facilitate the integration of the ARG from the plasmid to the more stable chromosome during transmission. 10o the best of our knowledge, this represents the first documented occurrence of both bla NDM-1 and mcr-1 in E. coli strains isolated from public trash cans, signifying the dissemination of strains carrying mcr-1 and bla NDM-1 beyond clinical and animal settings into the public domain, thereby significantly augmenting the risk to public health security.This underscores the imperative for continuous surveillance of polymyxin-resistant and carbapenem-resistant microorganisms by employing a 'One Health' approach to mitigate the proliferation of antibiotic-resistant bacteria.